This invention concerns heterocyclic derivatives which are useful in inhibiting oxido-squalene cyclase, processes for their preparation and pharmaceutical compositions containing them. The present invention is also concerned with heterocyclic derivatives capable of inhibiting cholesterol biosynthesis and hence in lowering cholesterol levels in blood plasma. The present invention also relates to methods of using such heterocyclic derivatives in treating or preventing diseases and medical conditions such as hypercholesterolemia, atherosclerosis and other medical conditions associated with elevated cholesterol levels.
There is evidence that high serum cholesterol levels are an important risk factor in coronary heart disease and associated diseases such as atherosclerosis and ischaemic heart disease. As a result there has been a great deal of interest in finding ways of lowering cholesterol levels in blood plasma. Although it is possible to obtain some reduction by means of diet, only modest reductions are obtained by controlling the dietary intake of cholesterol. Consequently, there is a need for therapeutic approaches to reducing cholesterol levels.
Several different classes of compounds have been reported which are able to lower cholesterol levels in blood plasma. For example agents which inhibit the enzyme HMGCoA reductase, an enzyme essential for the production of cholesterol, have been reported to reduce levels of serum cholesterol. Illustrative of this class of compounds is the HMGCoA reductase inhibitor known as lovastatin which is disclosed in U.S. Pat. No. 4,231,938. Other agents which are reported to lower serum cholesterol include those which act by complexing with bile acids in the intestinal system and which are hence termed xe2x80x9cbile acid sequestrantsxe2x80x9d. It is believed that these agents lower cholesterol levels indirectly by sequestering bile acids within the intestinal tract resulting in lower levels of bile acid circulating in the enteroheptatic system. Replacement of bile acids, which is synthesised in the liver from cholesterol, is promoted. This in turn results in an upregultion of the hepatic LDL cholesterol receptor and in a lowering of circulating blood cholesterol levels.
The biosynthesis of cholesterol is a complex process which will be considered here as three principal stages, namely 1) the conversion of acetic acid to mevalonic acid 2) the conversion of mevalonic acid to squalene and 3) the conversion of squalene to cholesterol. In the last stage, squalene is first converted into 2,3-oxido-squalene and then to lanosterol. Lanosterol is then converted to cholesterol through a number of enzymatic steps.
The conversion of 2,3-oxido-squalene to lanosterol is a key step in the biosynthesis of cholesterol. This conversion is catalysed by the enzyme oxido-squalene cyclase. It follows that inhibition of this enzyme decreases the amount of lanosterol available for conversion to cholesterol. Consequently, inhibition of oxido-squalene cyclase should interupt cholesterol biosynthesis and give rise to a lowering of cholesterol levels in blood plasma.
The present invention is based on the discovery that certain heterocyclic derivatives are inhibitors of oxido-squalene cyclase and are hence useful in treating diseases and medical conditions in which inhibition of oxido-squalene cyclase is desirable.
According to the present invention there is provided a compound of formula I (set out on a seperate sheet following the examples together with the other formulae referred to herein), or a pharmaceutically acceptable salt thereof, wherein:
G is selected from CH or N;
R1 is selected from hydrogen, halogeno, (1-6C)alkyl, halogeno(1-6C)alkyl, cyano, nitro, (1-6C)alkoxycarbonyl, and NR3R4 wherein R3 and R4 are independently selected from hydrogen and (1-6C)alkyl, and wherein upto 3 R1 groups may be present:
T1 is selected from CH or N;
T2 and T3 are independently selected from N and CR, wherein R is selected from hydrogen, hydroxyl and (C1-4)alkyl and wherein either ring containing T2 or T3 is optionally substituted with an oxo group;
R2 is selected from hydrogen or (1-4C)alkyl;
Q is selected from SO2, CO and CH2;
Ar is selected from a five or six membered heterocycle containing up to 3 heteroatoms selected from nitrogen, oxygen and sulphur, phenyl, phenyl (2-6C)alkenyl and naphthyl in which any Ar group is optionally substituted by one or more substituents selected from (1-6C)alkyl, halogeno, halogeno (1-6C)alkyl, (1-6C)alkoxy, (1-6C)alkoxycarbonyl, cyano, (1-6C)alkylamido, nitro, NR3R4 wherein R3 and R4 are independently selected from hydrogen and (1-4C)alkyl; provided that both T2 and T3 are not N and that when T2 is CR then T1 is not CH.
The compounds of the present invention are oxido-squalene cyclase inhibitors and hence possess the property of inhibiting cholesterol biosynthesis. Provided as a further feature of the invention is compounds of formula (I), or a pharmaceutically acceptable salt, for use in medical therapy. There is also provided the use of a compound of formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for treating diseases or medical conditions in which inhibition of oxidosqualene cyclase is desirable. There is also provided the use of a compound of formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for inhibiting cholesterol biosynthesis.
Thus according to a further feature of the present invention there is provided a method of inhibiting oxido-squalene cyclase in a warm-blooded animal (such as man) requiring such treatment, which method comprises adminstering to said animal an effective amount of a compound of formula I, or a pharmaceutically-acceptable salt thereof. In particular, the present invention provides a method of inhibiting cholesterol biosynthesis, and more particularly to a method of treating hypercholesterolemia and atheromatous vascular degeneration (such as atherosclerosis).
The compounds of the present invention are useful in treating diseases or medical conditions in which inhibition of oxido-squalene cyclase is desirable, for example those in which a lowering of the level of cholesterol in blood plasma is desirable. In particular, the compounds of the present invention are useful in treating hypercholesterolemia and/or ischaemic diseases associated with atheromatous vascular degeneration such as atherosclerosis.
Thus the present invention also provides the use of a compound of formula I, or a pharmaceutically-acceptable salt thereof, for the manufacture of a medicament for treating diseases or medical conditions in which a lowering of the level of cholesterol in blood plasma is desirable (such as hypercholesterolemia and atherosclerosis).
In particular, the compounds of the present invention are potentially useful in inhibiting cholesterol biosynthesis in man and hence in treating the above-mentioned medical conditions in man.
It will be understood that when compounds of formula I contain a chiral centre, the compounds of the invention may exist in, and be isolated in, optically active or racemic form. The invention includes any optically active or racemic form of a compound of formula I which possesses the beneficial pharmacological effect of inhibiting oxido-squalene cyclase. The synthesis of optically active forms may be carried out by standard techniques of organic chemistry well known in the art, for example by, resolution of a racemic form, by synthesis from optically active starting materials or by asymmetric synthesis. It will be appreciated that certain compounds of formula I may exist as geometrical isomers. The invention includes any geometrical isomer of a compound of formula I which possesses the beneficial pharmacological effect of inhibiting oxido-squalene cyclase.
It will also be understood that certain compounds of the present invention may exist in solvated, for example hydrated, as well as unsolvated forms. It is to be understood that the present invention encompasses all such solvated forms which possess the property of inhibiting oxido-squalene cyclase.
It is also to be understood that generic terms such as xe2x80x9calkylxe2x80x9d include both the straight chain and branched chain groups such as butyl and tert-butyl. However, when a specific term such as xe2x80x9cbutylxe2x80x9d is used, it is specific for the straight chain or xe2x80x9cnormalxe2x80x9d butyl group, branched chain isomers such as xe2x80x9ct-butylxe2x80x9d being referred to specifically when intended.
Particularly preferred compounds of formula are those wherein:
(i) Q is SO2 or CO, preferably SO2;
(ii) Ar is phenyl, naphthyl, thiophenyl, styryl or pyridyl, preferably phenyl or naphthyl, preferably phenyl;
(iii) R1 is hydrogen or (1-6C)alkyl;
(iv) R2 is hydrogen;
(v) substituents on the heterocyclic rings containing T2 and T3 are (1-6C)alkyl or (2-6C)alkenyl, preferably (1-6C)alkyl, preferably rings containing T2 and T3 are unsubstituted;
(vi) when Ar is substituted by halogeno, halogeno(1-6C)alkyl, (1-6C)alkoxy;
(vii) Ar is unsubstituted phenyl;
Particular values for Ar when a 5- or 6-membered heteroaryl moiety is present are furyl, thienyl, pyrrolyl, pyridyl and, preferably thienyl or pyridyl.
More particularly preferred embodiments are defined below, where substituents are not described they are as defined above.
(i) T1=N, T3=N and Ar is optionally substituted phenyl;
(ii) T1=N, T2=N and Ar is optionally substituted phenyl;
(iii) T1=CH, T3=N and Ar is optionally substituted phenyl;
(iv) T1=CH, T2=N and Ar is optionally substituted phenyl;
Compounds of particular interest include those described in the accompanying examples and their pharmaceutically acceptable salts.
The compounds of formula I and their pharmaceutically acceptable salts may be prepared by processes known to be applicable to the preparation of structurally related compounds. These procedures are illustrated by the following representative processes in which the various groups and radicals such as R1, R2, G, T1, T2, T3, Q and Ar are as defined above (unless stated otherwise), and are provided as a further feature of the present invention. In cases where the compounds contain a group such as an amino, hydroxy, or carboxy group, this group may be protected using a conventional protecting group which may be removed when desired by conventional means. The compounds of formula 1 may be prepared in accordance with the following alternative procedures:
(a) reacting an amine of formula IIA or IIB with a compound of formula III, wherein L is a leaving group or atom (for example chloro or bromo) in the presence of a base, for example triethylamine or pyridine. The reaction is preferably carried out in a suitable inert solvent such as methylene dichloride, tetrahydrofuran or water and at a temperature range of from xe2x88x9220 to 50xc2x0 C., conveniently at or near ambient temperature;
(b) reducing a compound of formula IV. Suitable agents to effect the reduction of compounds of formula IV include borane complexes, such as borane-dimethylsulphide, and complex metal hydrides, such as aluminium lithium hydride. The reaction is preferably carried out in a suitable inert solvent, such as tetrahydrofuran or diethylether, and at a temperature range of from 0 to 25xc2x0 C.;
(c) reacting a compound of formula V, where T1 is N, with a compound of formula VI, in which Z is a displaceable group, such as halo (for example chloro). The reaction is preferably carried out in the presence of a base, such as sodium hydrogencarbonate, triethylaamine or pyridine and in a suitable inert solvent, such as an alcohol (for example ethanol), methylene chloride or tetrahydrofuran, at a temperature range of from 20 to 120xc2x0 C.;
Compounds of formula IIA may be prepared by reductive amination, reacting a compound of formula VII, where T2 is N and X is H, with a compound of formula VIII, where T3 is CH, Y is CHO or COR2 and P is a protecting group (for example benzyloxycarbonyl), and subsequently effecting removal of the protecting group. Alternatively compounds of formula IIA may be prepared by reductive amination, reacting a compound of formula VII, where T2 is CH and X is CHO or COR2, with a compound of formula VIII, where T3 is N, Y is H and P is a protecting group (for example benzyloxycarbonyl), and subsequently effecting removal of the protecting group. Each reductive amination reaction is preferably carried out in a suitable inert solvent, such as an alcohol (for example methanol), and at a temperature range of 0xc2x0 C. to ambient temperature, and in the presence of a suitable reducing agent, such as a borane complex, for example sodiumcyanoborane hydride.
Alternatively compounds of formula IIA may be prepared by reacting a compound of formula X, wherein P is a protecting group (for example benzyloxycarbonyl), with a compound of formula VI in an analogous manner as described in method c) above.
Compounds of formula IIB may be prepared by reacting a compound of formula IX with a compound of formula VIII, wherein T3 is N, Y is H and P1 is a protecting group, and subsequently effecting removal of the protecting group. Preferable the reaction is carried out in a suitable inert solvent such as aqueous alcohol (for example aqueous propanol) at a temperature range of from 60-140xc2x0 C. Removal of the protecting group is by reaction corresponding to the particular protecting group used (for example for benzyloxycarbonyl by hydrogenation in the presence of a catalyst such as palladium-on-carbon).
Compounds of formula IV may be prepared in accordance with the procedures described in WO 97/06802.
Compounds of formula VII, where X is CHO, and compounds of formula VIII, where Y is CHO, may be prepared by reduction of the corresponding Weinreb amide, suitably using a complex metal hydride such as lithium aluminium hydride.
Compounds of formula IX may be prepared by reacting a compound of formula VII, where T2 and X form a carbonyl group, to form the epoxide. Epoxide formation is effected by trimethyl sulpoxonium iodide (coreyylide).
A suitable protecting group for an amino or alkylamino group is, for example, an acyl group for example an alkanoyl group such as acetyl, an alkoxycarbonyl group, for example a methoxycarbonyl, ethoxycarbonyl or tert-butoxycarbonyl group, an arylmethoxycarbonyl group, for example benzyloxycarbonyl, or an aroyl group, for example benzoyl. The deprotection conditions for the above protecting groups necessarily vary with the choice of protecting group. Thus, for example, an acyl group such as an alkanoyl or alkoxycarbonyl group or an aroyl group may be removed for example, by hydrolysis with a suitable base such as an alkali metal hydroxide, for example lithium or sodium hydroxide. Alternatively an acyl group such as a tert-butoxycarbonyl group may be removed, for example, by treatment with a suitable acid as hydrochloric, sulphuric or phosphoric acid or trifluoroacetic acid and an arylmethoxycarbonyl group such as a benzyloxycarbonyl group may be removed, for example, by hydrogenation over a catalyst such as palladium-on-carbon, or by treatment with a Lewis acid for example boron tris(trifluoroacetate). A suitable alternative protecting group for a primary amino group is, for example, a phthaloyl group which may be removed by treatment with an alkylamine, for example dimethylaminopropylamine, or with hydrazine.
A suitable protecting group for a hydroxy group is, for example an acyl group, for example an alkanoyl group such as acetyl, an aroyl group, for example benzoyl, or an arylmethyl group, for example benzyl. The deprotection conditions for the above protecting groups will necessarily vary with the choice of protecting group. Thus, for example, an acyl group such as an alkanoyl or an aroyl group may be removed, for example, by hydrolysis with a suitable base such as an alkali metal hydroxide, for example lithium or sodium hydroxide. Alternatively an arylmethyl group such as a benzyl group may be removed, for example, by hydrogenation over a catalyst such as palladium-on-carbon.
A suitable protecting group for a carboxy group is, for example, an esterifying group, for example a methyl or an ethyl group which may be removed, for example, by hydrolysis with a base such as sodium hydroxide, or for example a tert-butyl group which may be removed, for example, by treatment with an acid, for example an oreganic acid such as trifluoroacetic acid, or for example a benzyl group which may be removed, for example, by hydrogenation over a catalyst such as palladium-on-carbon.
As mentioned above, it will be appreciated that in some of the reactions mentioned herein it may be necessary/desirable to protect any sensitive groups in the compounds. The instances where protection is necessary or desirable and suitable methods for protection are known to those skilled in the art. Thus, if reactants include groups such as amino, carboxy or hydroxy it may be desirable to protect the group in some of the reactions mentioned herein. The protecting groups may be removed at any convenient stage in the synthesis using conventional techniques well known in the chemical art.
It will also be appreciated that certain of the various optional substituents in the compounds of the present invention may be introduced by standard aromatic substitution reactions or generated by conventional functional group modifications either prior to or immediately following the processes mentioned above, and as such are included in the process aspect of the invention. Such reactions and modifications include, for example, introduction of a substituent by means of an aromatic substitution reaction, reduction of substituents, alkylation of substituents and oxidation of substituents. The reagents and reaction conditions for such procedures are well known in the chemical art. Particular examples of aromatic substitution reactions include the introduction of a nitro group using concentrated nitric acid, the introduction of an acyl group using, for example, an acylhalide and Lewis acid (such as aluminium trichloride) under Friedel Crafts conditions; the introduction of an alkyl group using an alkyl halide and Lewis acid (such as aluminium trichloride) under Fiedel Crafts conditions; and the introduction of a halogeno group. Particular examples of modifications include the reduction of a nitro group to an amino group by for example, catalytic hydrogenation with a nickel catalyst or treatment with iron in the presence of hydrochloric acid with heating; oxidation of alkylthio to alkylsulphinyl or alkylsulphonyl.
When a pharmaceutically-acceptable salt of a compound of the formula I is required, it may be obtained, for example, by reaction of said compound with the appropriate acid (which affords a physiologically acceptable anion), or with the appropriate base (which affords a physiologically acceptable cation), or by any other conventional salt formation procedure.
When an optically active form of a compound of the formula I is required, it may be obtained, for example, by carrying out one of the aforesaid procedures using an optically active starting material or by resolution of a racemic form of said compound using a conventional procedure.
As mentioned previously, the compounds of the formula I (and their pharmaceutically-acceptable salts) are inhibitors of the enzyme oxido-squalene cyclase. Thus, the compounds of the present invention are capable or inhibiting cholesterol biosynthesis and hence in lowering choleserol levels in blood plasma.
The beneficial pharmacological properties of the compounds of the present invention may be demonstrated using one or more of the following techniques.
(a) In vitro Test to Measure Inhibition of Oxido-squalene Cyclase
This test measures the inhibition of microsomal oxido-squalene cyclase in vitro by compounds at set concentrations in the incubation medium.
Microsomes are prepared from rat liver according to methods known in the art, for example, the method described in published European Patent Application No 324,421 and stored in liquid nitrogen prior to assay. Assay vials are kept at 37xc2x0 C. throughout the incubation. The microsomes typically contain 15-20 mg of protein per ml of microsomes. For 7.4.
Phosphate buffered Tween 80 (polyoxyethylene sorbitan monolaurate) is prepared by adding 0.1 g tween 80 to 100 ml of 50 mM phosphate buffer.
A stock solution of oxido-squalene is made up as a solution in ethanol (0.65 mg. ml.xe2x88x921). 18 xcexcl of radio-labelled oxido-squalene (1 xcexcCi.mlxe2x88x921) is evaporated to dryness under a stream of nitrogen and redissolved in 1 ml of ethanol and 1 ml of the stock solution of oxido-squalene is added.
The test compound is dissolved in dimethyl sulphoxide to give a 10xe2x88x924M stock solution. Dilutions are made from the stock to give 10xe2x88x925M, 10xe2x88x926M etc.
Phosphate buffered Tween 80 (28 xcexcl) is placed in 5 ml disposable plastic vials and 4 xcexcl of the solution of the test compound is added and mixed well. An aliquot of the oxido-squalene mix (15 xcexcl) is added and the vials pre-incubated for 10 minutes at 37xc2x0 C. A portion of the microsomes (14.6 xcexcl) are then added and incubated for a further 1 hour. The reaction is stopped by the addition of 315 xcexcl of a mixture of 16% KOH in 20% ethanol.
The samples are then placed in a water bath at 80xc2x0 C. for 2 hours to saponify. At the end of this process water (630 xcexcl) is added followed by hexane (5 ml). The samples are tumble mixed for 5 minutes and then centrifuged. The hexane phase is removed and evaporated under nitrogen. The samples are then reconstituted in 300 xcexcl of a 80:20 mixture of a acetonitrile:isopropyl alcohol. The samples are then chromatographed using a Hichrom 30DsS1 column with an isocratic elution using a 95:5 mixture of acetonitrile:isopropyl alcohol and a flow rate of 1 ml.minxe2x88x921. The output from the UV detector is connected to a radio-chemical detector to visualise radiolabelled sterols. Reaction rate is measured as the conversion of oxido-squalene to lanosterol, and the effects of test compounds are expressed as an inhibition of this process.
(b) In vivo Test to Measure Inhibition of Oxido-squalene Cyclase
The ability of a compound to inhibit oxido-squalene cyclase and/or inhibit cholesterol biosynthesis may be assessed by a routine laboratory procedure carried out in the rat. The test involves administration of the compound to rats on a reversed lighting regimen. Female rats (35-55 g) are housed in reverse lighting conditions (red light from 0200 h-1400 h) for a period of about 2 weeks prior to test. Animals are allowed free access to chow and drinking water throughout this period. At test, animals should weigh 100-140 g. The rats are dosed orally with the compound (typically 10-50 mg/kg) formulated in apolyethylene glycol/hydroxypropylmethyl cellulose mix. After 1 hour the rats are given triturated sodium mevalonate (15 xcexcCi/kg) intraperitoneally. Two hours after administration of the compound the rats are terminated and a piece of liver removed and weighed. The tissue is saponified at 80xc2x0 C. for 2 hours in an ethanolic/potassium hydroxide solution (80% w/v aqueous KOH diluted 1:10 with ethanol). Water (2 ml) is added and the mixture extracted wiht iso-hexane (2xc3x975 ml). The organic extracts are combined, evaporated to dryness under a stream of nitrogen and the residue is dissolved in a mixture of acetonitrile/iso-propanol (300 xcexcl). An aliquot (200 xcexcl) of this solution is loaded onto a HPLC column to separate the sterols. The radio-label content of each fraction is assessed using a radio chemical flow detector. Inhibitors of oxido squalene cyclase are classed as those compounds which caused a build up of substrate and a concomitant disappearance of cholesterol and its precursors. ED50 values are generated in the usual manner.
As mentioned previously, the compounds of the present invention are inhibitors of oxido-squalene cvclase and hence possess the property of inhibiting cholesterol biosynthesis. Thus the compounds of the present invention will be useful in treating diseases or medical conditions in which an inhibition of choleserol biosynthesis or lowering of cholesterol levels in blood plasma is desirable, for example, hypercholesterolemia and/or ischaemic diseases associated with atheromatous vascular degeneration such as atherosclerosis.
When used in the treatment of diseases and medical conditions such as those mentioned above it is envisaged that a compound of formula I, or a pharmaceutically acceptable salt thereof, will be administered orally intravenously, or by some other medically acceptable route so that a dose in the general range of, for example, 0.01 to 10 mg per kg body weight is received. However it will be understood that the precise dose administered will necessarily vary according to the nature and severity of the disease, the age and sex of the patient being treated and the route of administration.
In general, the compounds of formula I, or a pharmaceutically-acceptable salt thereof, will usually be administered in the form of a pharmaceutical composition, that is together with a pharmaceutically acceptable diluent or carrier, and such a composition is provided as a further feature of the present invention.
A pharmaceutical composition of the present invention may be in a variety of dosage forms. For example, it may be in the form of tablets, capsules, solutions or suspensions for oral administration, in the form of suppository for rectal administration; in the form of a sterile solution or suspension for parenteral administration such as by intravenous or intramuscular injection.
A composition may be obtained by conventional procedures using pharmaceutically acceptable diluents and carriers well known in the art. Tablets and capsules for oral administration may conveniently be formed with a coating, such as an enteric coating (for example, one based on cellulose acetate phthalate), to minimise dissolution of the active ingredient of formula I, or a phannaceutically-acceptable salt thereof, in the stomach or to mask unpleasant taste.
The compounds of the present invention may, if desired, be administered together with (or sequentially to) one or more other pharmacological agents known to be useful in the treatment of cardiovascular disease, for example, together with agents such as HMG-CoA reductase inhibitors, bile acid sequestrants, other hypocholesterolaemic agents such as fibrates, for example gemfibrozil, and drugs for the treatment of coronary heart disease.
As inhibitors of oxido-squalene cyclase, the compounds of the present invention may also find utility as antifungal agents, and so the present invention also provides a method of inhibiting cholesterol biosynthesis in fungi. In particular the present invention provides a method of treating fungal infections which comprises administration to a warm blooded animal, such as man, in need of such treatment an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof. When used in this way the compounds of the present invention may, in addition to the formulations mentioned above, be adapted for topical administration and such a composition is provided as a further feature of the present invention. Such compositions may be in a variety of forms, for example creams or lotions.
The invention will now be illustrated by the following non-limiting Examples in which, unless otherwise stated:
(i) evaporations were carried out by rotary evaporation in vacuo;
(ii) operations were carried out at room temperature, that is in the range 18-26xc2x0 C.;
(iii) flash column chromatography or medium pressure liquid chromatography (MPLC) was performed on silica gel (Merck Kieselgel Art.9385, obtained from E Merck, Darmstadt, Germany);
(iv) yields are given for illustration only and are not necessarily the maximum attainable by diligent process development;
(v) proton NMR spectra were normally determined at 200 MHz using tetramethylsilane (TMS) as an internal standard, and are expressed as chemical shifts (delta values) obtained in DMSO-d6 (unless stated otherwise) in parts per million relative to TMS using conventional abbreviations for designation of major peaks: s, singlet, m, multiplet; t, triplet; br, broad; d, doublet;
(vi) all end-products were characterised by microanalysis, NMR and/or mass spectroscopy.